Cell Culture BASICS


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Cell Culture BASICS

  2. 2. What is Cell Culture? <ul><li>In vitro culture (maintain and/or proliferate) of cells, tissues or organs </li></ul><ul><li>Types of tissue culture </li></ul><ul><ul><li>Organ culture </li></ul></ul><ul><ul><li>Tissue culture </li></ul></ul><ul><ul><li>Cell culture </li></ul></ul>
  3. 3. Organ Culture <ul><li>The entire embryos or organs are excised from the body and culture </li></ul><ul><li>Advantages </li></ul><ul><ul><li>Normal physiological functions are maintained. </li></ul></ul><ul><ul><li>Cells remain fully differentiated. </li></ul></ul><ul><li>Disadvantages </li></ul><ul><ul><li>Scale-up is not recommended. </li></ul></ul><ul><ul><li>Growth is slow. </li></ul></ul><ul><ul><li>Fresh explantation is required for every experiment. </li></ul></ul>
  4. 4. Tissue Culture <ul><li>Fragments of excised tissue are grown in culture media </li></ul><ul><li>Advantages </li></ul><ul><ul><li>Some normal functions may be maintained. </li></ul></ul><ul><ul><li>Better than organ culture for scale-up but not ideal. </li></ul></ul><ul><li>Disadvantages </li></ul><ul><ul><li>Original organization of tissue is lost. </li></ul></ul>
  5. 5. Cell Culture <ul><li>Tissue from an explant is dispersed, mostly enzymatically, into a cell suspension which may then be cultured as a monolayer or suspension culture. </li></ul><ul><li>Advantages </li></ul><ul><ul><li>Development of a cell line over several generations </li></ul></ul><ul><ul><li>Scale-up is possible </li></ul></ul><ul><li>Disadvantages </li></ul><ul><ul><li>Cells may lose some differentiated characteristics. </li></ul></ul>
  6. 6. Why do we need Cell culture? <ul><li>Research </li></ul><ul><ul><li>To overcome problems in studying cellular behavior such as: </li></ul></ul><ul><ul><ul><li>confounding effects of the surrounding tissues </li></ul></ul></ul><ul><ul><ul><li>variations that might arise in animals under experimental stress </li></ul></ul></ul><ul><ul><li>Reduce animal use </li></ul></ul><ul><li>Commercial or large-scale production </li></ul><ul><ul><li>Production of cell material: vaccine, MAbs, hormone etc which are impossible to produce synthetically. </li></ul></ul>
  7. 7. Advantages of Cell culture <ul><li>Advantages: </li></ul><ul><ul><li>Absolute control of physical environment </li></ul></ul><ul><ul><li>Homogeneity of sample </li></ul></ul><ul><ul><li>Less compound needed than in animal models </li></ul></ul><ul><li>Disadvantages: </li></ul><ul><ul><li>Hard to maintain </li></ul></ul><ul><ul><li>Only grow small amount of tissue at high cost </li></ul></ul><ul><ul><li>Dedifferentiation </li></ul></ul><ul><ul><li>Instability, aneuploidy </li></ul></ul>
  8. 8. Characteristics of Animal Cell Culture <ul><li>Nutritionally demanding </li></ul><ul><li>Sensitive to shear and extremes of osmolality </li></ul><ul><li>Doubling time 12 to 48 hrs </li></ul><ul><li>Cell Density </li></ul>
  9. 9. Current Choices of Host Cells in Biotech Bacteria Cells Yeast Transgenic Animals Transgenic Plants Animal Cells
  10. 10. Comparison of Monoclonal Antibody Produced from CHO & Transgenic Goats Assumption Annual Yield (Kg/yr) Batch Yield (grams/L) 60 goat herd 350 L/animal year 40 5.0 Transgenic Goats Grange Castle 6 X 12,500 L Bioreactors 4000 3.4 CHO Bioreactor
  11. 11. The Majority of Biotech Products on the Market Are Made in Animal Cells
  12. 12. Comparison of Animal and Microbial Culture 10 6 cells/mL 10 9 -10 10 cells/mL Growth density 10 5 cells/mL 1 cell Seeding density 10000-100000 nm 100-2000 nm Size Very susceptible Less affected Environmental FX Key for buffering Sometimes CO 2 Requirement Complex Usually simple Nutritional Rqmt Low High O 2 Requirement 1-5% per hour 10-50% per hour Growth Rate Present Present Cell membrane Generally absent Generally present Cell wall Animal Cells Microbes Features
  13. 13. Types of Animal Cell culture <ul><li>Primary Cultures </li></ul><ul><ul><li>Derived directly from excised tissue and cultured either as </li></ul></ul><ul><ul><ul><li>Outgrowth of excised tissue in culture </li></ul></ul></ul><ul><ul><ul><li>Dissociation into single cells (by enzymatic digestion or mechanical dispersion) </li></ul></ul></ul><ul><ul><li>Advantages: </li></ul></ul><ul><ul><ul><li>usually retain many of the differentiated characteristics of the cell in vivo </li></ul></ul></ul><ul><ul><li>Disadvantages: </li></ul></ul><ul><ul><ul><li>initially heterogeneous but later become dominated by fibroblasts. </li></ul></ul></ul><ul><ul><ul><li>the preparation of primary cultures is labor intensive </li></ul></ul></ul><ul><ul><ul><li>can be maintained in vitro only for a limited period of time. </li></ul></ul></ul>
  14. 14. Types of Cell culture <ul><li>Continuous Cultures </li></ul><ul><ul><li>derived from subculture (or passage, or transfer) of primary culture </li></ul></ul><ul><ul><ul><li>Subculture = the process of dispersion and re-culture the cells after they have increased to occupy all of the available substrate in the culture </li></ul></ul></ul><ul><ul><li>usually comprised of a single cell type </li></ul></ul><ul><ul><li>can be serially propagated in culture for several passages </li></ul></ul><ul><ul><li>There are two types of continuous cultures </li></ul></ul><ul><ul><ul><li>Cell lines </li></ul></ul></ul><ul><ul><ul><li>Continuous cell lines </li></ul></ul></ul>
  15. 15. Types of continuous culture <ul><ul><li>Cell lines </li></ul></ul><ul><ul><ul><li>finite life, senesce after approximately thirty cycles of division </li></ul></ul></ul><ul><ul><ul><li>usually diploid and maintain some degree of differentiation. </li></ul></ul></ul><ul><ul><ul><li>it is essential to establish a system of Master and Working banks in order to maintain such lines for long periods </li></ul></ul></ul>
  16. 16. Types of continuous culture <ul><ul><li>Continuous cell lines </li></ul></ul><ul><ul><ul><li>can be propagated indefinitely </li></ul></ul></ul><ul><ul><ul><li>generally have this ability because they have been transformed </li></ul></ul></ul><ul><ul><ul><ul><li>tumor cells. </li></ul></ul></ul></ul><ul><ul><ul><ul><li>viral oncogenes </li></ul></ul></ul></ul><ul><ul><ul><ul><li>chemical treatments. </li></ul></ul></ul></ul><ul><ul><ul><li>the disadvantage of having retained very little of the original in vivo characteristics </li></ul></ul></ul>
  17. 17. Immortality of continuous culture <ul><ul><li>Telomeres lose about 100 base pairs from their telomeric DNA at each mitosis which impose a finite life span on cells after 125 mitotic divisions, the telomeres would be completely gone </li></ul></ul><ul><ul><li>Immortal cells maintain telomere length with the aid of an enzyme Telomerase </li></ul></ul><ul><ul><ul><li>adds telomere repeat sequences to the 3' end of DNA strands </li></ul></ul></ul><ul><ul><ul><li>help complete the synthesis of the &quot;incomplete ends&quot; </li></ul></ul></ul>
  18. 18. Cell Culture Morphology <ul><li>Morphologically cell cultures take one of two forms: </li></ul><ul><ul><li>Anchorage independent cells (Suspension culture) </li></ul></ul><ul><ul><li>Anchorage dependent cells (Adherent Culture) </li></ul></ul>
  19. 19. Cell Culture Morphology <ul><li>Morphologically cell cultures take one of two forms: </li></ul><ul><ul><li>growing in suspension (as single cells or small free-floating clumps) </li></ul></ul><ul><ul><ul><li>are able to survive and proliferate without attachment to the culture vessel </li></ul></ul></ul><ul><ul><ul><li>cells from blood, spleen, bone marrow, etc </li></ul></ul></ul><ul><ul><ul><li>advantage: large numbers, ease of harvesting </li></ul></ul></ul><ul><ul><li>growing as a monolayer that is attached to any surface. </li></ul></ul><ul><ul><ul><li>grow in monolayer, attached to the surfaces of the culture vessels </li></ul></ul></ul><ul><ul><ul><li>from ectodermal or endodermal embryonic cells, e.g. fibroblasts, epithelial cells </li></ul></ul></ul><ul><ul><ul><li>various shapes but generally are flat (rounded in suspension) </li></ul></ul></ul><ul><ul><ul><li>Advantage: spread on surfaces such as coverslips, easy for microscopy or other functional assays </li></ul></ul></ul>
  20. 20. Development of Cell Lines
  21. 21. Bioreactor <ul><li>A bioreactor may refer to any device or system that supports a biologically active environment. </li></ul>
  22. 22. Requirements for a bioreactor for animal cell culture <ul><ul><li>1) well-controlled environment (T, pH, DO, nutrients, and wastes) </li></ul></ul><ul><ul><li>2) supply of nutrients </li></ul></ul><ul><ul><li>3) gentle mixing (avoid shear damage to cells) </li></ul></ul><ul><ul><li>4) gentle aeration (add oxygen slowly to the culture medium, but avoid the formation of large bubbles which can damage cells on contact). </li></ul></ul><ul><ul><li>5) removal of wastes </li></ul></ul>
  23. 23. Scale-up <ul><li>Start with small volume reactors </li></ul><ul><ul><li>T flasks, shaker flasks (5-25 mL) </li></ul></ul><ul><li>Intermediate scale </li></ul><ul><ul><li>Small, highly controlled bioreactors (1-5 L) </li></ul></ul><ul><li>Production scale </li></ul><ul><ul><li>Large reactors (20-1,000 L) </li></ul></ul>
  24. 24. Reactor types <ul><li>Tissue flasks </li></ul><ul><ul><li>Easy to use for small scale </li></ul></ul><ul><li>Cell factories </li></ul><ul><ul><li>Production of large numbers of cells </li></ul></ul><ul><ul><li>Labor intensive </li></ul></ul><ul><li>Roller bottles </li></ul><ul><ul><li>Good control of gas phase </li></ul></ul><ul><ul><li>Labor intensive </li></ul></ul><ul><li>Hollow fiber systems </li></ul><ul><ul><li>High cell densities, good oxygenation </li></ul></ul><ul><ul><li>Difficult to remove cells </li></ul></ul><ul><li>Spinner flasks </li></ul><ul><ul><li>Mimic a traditional stirred tank reactor </li></ul></ul>
  25. 25. Types on the basis of mode of operation <ul><li>Batch </li></ul><ul><li>Fed Batch </li></ul><ul><li>Continuous </li></ul>
  26. 26. Batch Culture <ul><li>A closed culture system which contains an initial, limited amount of nutrient. The inoculated culture will pass through a number of phases following a growth curve. The growth curve contains four distinct regions as </li></ul><ul><ul><li>Lag Phase </li></ul></ul><ul><ul><li>Exponential Phase </li></ul></ul><ul><ul><li>Stationary Phase </li></ul></ul><ul><ul><li>Death Phase </li></ul></ul>
  27. 27. Lag Phase <ul><li>The first major phase of growth in a batch bioreactor </li></ul><ul><li>A period of adaptation of the cells to their new environment </li></ul><ul><li>Minimal increase in cell density </li></ul><ul><li>May be absent in some Bioreactors (depends on seed culture) </li></ul>
  28. 28. Exponential Phase <ul><li>Also known as the logarithmic growth phase </li></ul><ul><li>Cells have adjusted to their new environment The cells are dividing at a constant rate resulting in an exponential increase in the number of cells present. This is known as the specific growth rate and is represented mathematically by first order growth rate </li></ul><ul><li>dX = (μ – kd) X </li></ul><ul><li>dt </li></ul><ul><li>where X is the cell concentration, </li></ul><ul><li>μ is the cell growth rate </li></ul><ul><li>kd is the cell death rate. </li></ul><ul><li>The cell death rate is sometimes neglected if it is considerably smaller than the cell growth rate. </li></ul>
  29. 29. Exponential Phase <ul><li>Cell growth rate is often substrate limited, as depicted in the figure to limited the right. </li></ul><ul><li>The growth curve is well represented by Monod batch kinetics, which is mathematically depicted on the following slide. </li></ul>
  30. 30. Exponential Phase <ul><li>Monod batch kinetics is represented mathematically in the following equation: </li></ul><ul><li>μ = μ max S </li></ul><ul><li> Ks+ S </li></ul><ul><li>where μ is the specific growth rate, μ max is the maximum specific growth rate, S is the growth limiting substrate concentration and Ks is the saturation constant which is equal to the substrate concentration that produces a specific growth rate equal to half the max specific growth rate </li></ul>
  31. 31. Exponential Phase <ul><li>For Primary Metabolite production conditions to extend the exponential phase accompanied by product excretion </li></ul><ul><li>For Secondary Metabolite production, conditions giving a short exponential phase and an extended production phase, or conditions giving a decreased growth rate in the log phase resulting in earlier secondary metabolitwe formation. </li></ul>
  32. 32. Stationary Phase <ul><li>The third major phase of microbial growth in a batch process occur when the number of cells dividing and dying is in equilibrium and can be the result of the following </li></ul><ul><ul><li>Depletion of one or more essential growth nutrients </li></ul></ul><ul><ul><ul><li>Primary metabolite, or growth associated, production stops </li></ul></ul></ul><ul><ul><ul><li>Secondary metabolite or non-growth associated, production may continue </li></ul></ul></ul><ul><ul><li>Accumulation of toxic growth associated by-products </li></ul></ul><ul><ul><li>Stress associated with the induction of a recombinant gene </li></ul></ul>
  33. 33. Death Phase <ul><li>The rate of cells dying is greater than the rate of cells dividing </li></ul><ul><li>represented mathematically by first order kinetics as following </li></ul><ul><li> dx = -k d X </li></ul><ul><li>dt </li></ul>
  34. 34. Batch Curve
  35. 35. Fed Batch Culture <ul><li>Types of Fed Batch Culture </li></ul><ul><ul><li>Intermittent Harvest </li></ul></ul><ul><ul><ul><li>Grow up the culture, harvest and refill with fresh medium </li></ul></ul></ul><ul><ul><li>Fed Batch Culture </li></ul></ul><ul><ul><li>Extended Fed Batch Culture </li></ul></ul><ul><ul><li>Fed Batch Culture with metabolic shift </li></ul></ul>
  36. 36. Intermittent Harvest <ul><li>In general, fed batch processes do not deviate significantly from batch cultures. </li></ul><ul><li>Cells are inoculated at a lower viable cell density in a medium that is usually very similar in composition to a typical batch medium. </li></ul><ul><li>Cells are allowed to grow exponentially with essentially no external manipulation until nutrients are somewhat depleted and cells are approaching the stationary growth phase. </li></ul>
  37. 37. Intermittent Harvest <ul><li>At this point, a portion of the cells and product are harvested, and the removed culture fluid is replenished with fresh medium </li></ul><ul><li>This process is repeated several times, as it allows for an extended production period. </li></ul>
  38. 38. Fed Batch Culture <ul><li>While cells are still growing exponentially, but nutrients are becoming depleted, concentrated feed medium (usually a 10-15 times concentrated basal medium) is added either continuously (as shown) or intermittently to supply additional nutrients, allowing for a further increase in cell concentration and the length of the production phase. </li></ul><ul><li>In contrast to an intermittent-harvest strategy, fresh medium is added proportionally to cell concentration without any removal of culture broth. </li></ul><ul><li>To accommodate the addition of medium, a fedbatch culture is started in a volume much lower than the full capacity of the bioreactor </li></ul>
  39. 40. Extended Fed Batch Culture <ul><li>Grow up the cells, then begin to feed concentrate of medium components, viability continues to decrease but cell and product concentrations continue to increase. </li></ul><ul><li>Can reach very high product and cell concentration. </li></ul>
  40. 41. Fed Batch Culture with Metabolic Shift <ul><li>In batch cultures and most fedbatch processes, lactate, ammonium, and other metabolites eventually accumulate in the culture broth over time, affecting cell growth, glycoform of the product and productivity. </li></ul><ul><li>Other factors, such as high osmolarity and accumulation of reactive oxygen species, are also growth inhibitory </li></ul>
  41. 42. Fed Batch Culture with Metabolic Shift <ul><li>After extended exposure to low glucose concentrations, cell metabolism is directed to a more efficient state, characterized by a dramatic reduction in the amount of lactate produced. Such a change in cell metabolism from the normally observed high lactate producing state to a much reduced lactate production state is often referred to as metabolic shift. </li></ul><ul><li>Very high cell concentrations and product titers were achieved in hybridoma cells. </li></ul>
  42. 43. Cell retention and perfusion <ul><ul><li>Characterized by the continuous addition of fresh nutrient medium and the withdrawal of an equal volume of used medium. </li></ul></ul><ul><li>Need of perfusion </li></ul><ul><ul><li>Product is unstable </li></ul></ul><ul><ul><li>Product concentration is low </li></ul></ul><ul><li>Perfusion technologies </li></ul><ul><ul><ul><li>Enhanced sedimentation </li></ul></ul></ul><ul><li> Conical settlers </li></ul><ul><li>Incline settlers </li></ul><ul><li>Lamellar settlers </li></ul><ul><ul><ul><li>Centrifugation </li></ul></ul></ul><ul><ul><ul><li>Spin filters </li></ul></ul></ul><ul><li> External </li></ul><ul><li>Internal </li></ul>
  43. 44. Perfusion Culture
  44. 45. Advantages of Perfusion Technology <ul><li>Better economics </li></ul><ul><li>High cell density </li></ul><ul><li>High productivity </li></ul><ul><li>Longer operation duration </li></ul><ul><li>Small fermenter size </li></ul><ul><li>flexibility </li></ul><ul><li>Fast start up in process development </li></ul><ul><li>Constant nutrient supply </li></ul><ul><li>Better controlled culture environment </li></ul><ul><li>Steady state operation </li></ul><ul><li>Ease of control </li></ul><ul><li>Better product quality </li></ul>
  45. 46. Disadvantages of Perfusion Technology <ul><li>Contamination risk </li></ul><ul><li>Equipment failure </li></ul><ul><li>Increased analytical costs </li></ul><ul><li>Long validation time </li></ul><ul><li>Potential regulatory/licensing issues </li></ul>
  46. 47. <ul><li>Thank you </li></ul>
  47. 48. <ul><li>Stirred Tank Bioreactor </li></ul><ul><li>Bubble Column Bioreactor </li></ul><ul><li>Air lift Bioreactor </li></ul><ul><li>Fluidized bed Bioreactor </li></ul><ul><li>Packed Bed Bioreactor </li></ul><ul><li>Flocculated Cell reactors </li></ul><ul><li>Wave </li></ul><ul><li>Hollow fiber </li></ul><ul><li>Perfusion </li></ul><ul><li>Encapsulation </li></ul>
  48. 49. McLimans' group developed the first &quot;spinner flasks&quot; in 1957. Present Model Original Model
  49. 50. Advantages of Spinner Flasks <ul><li>Easy </li></ul><ul><li>Visible </li></ul><ul><li>Cheap </li></ul><ul><li>Depyrogenation feasible </li></ul>
  50. 51. Disadvantages of Spinner Flasks <ul><li>Poor aeration </li></ul><ul><li>Impeller jams </li></ul><ul><li>Requires cleaning siliconizing & sterilization </li></ul><ul><li>High space requirements in incubator </li></ul>
  51. 52. Four Basic Bioreactor Designs <ul><ul><li>Stirred tank reactors (mechanical agitation for aeration) </li></ul></ul><ul><ul><li>Bubble column reactors (bubbling air into media for aeration) </li></ul></ul><ul><ul><li>Internal loop airlift reactors (air and media circulate together) </li></ul></ul><ul><ul><li>External loop airlift reactors </li></ul></ul>
  52. 53. Bioreactor Design Airlift Reactors Stirred Tank Reactor
  53. 54. Stirred Tank Bioreactor
  54. 55. Advantages of Stirred Tank Bioreactor <ul><li>Versatility </li></ul><ul><li>Multi-gas and pH control </li></ul><ul><li>Increased Capacity( 5 L to 500 L +) </li></ul>
  55. 56. Disadvantages of Stirred Tank Bioreactor <ul><li>Costly </li></ul><ul><li>Size (footprint)/ Weight </li></ul><ul><li>Preparation - siliconizing, cleaning, </li></ul><ul><li>Sterilization, depyrogenation </li></ul><ul><li>Maintenance -Chiller, parts, o-rings </li></ul>
  56. 57. Disposable Bioreactor <ul><ul><ul><ul><li>Can be scaled to at least 500 liters </li></ul></ul></ul></ul><ul><ul><ul><ul><li>A non-invasive agitation mechanism </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Easy to use </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Disposable, presterile, and biocompatible </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Well instrumented, and can be sampled </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Useful for suspension and adherent culture </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Suitable for GMP operation </li></ul></ul></ul></ul>
  57. 58. Wave Bioreactor
  58. 59. Wave Bioreactor
  59. 60. Wave-induced Agitation
  60. 61. Advantage of Wave Bioreactor <ul><li>DISPOSABLE BIOREACTOR CHAMBER . No cross-contamination, cleaning, sterilization or other validation headaches. </li></ul><ul><li>SEED PREPARATION </li></ul><ul><li>Seed culture can be prepared in the final system itself, i.e. batch can be started with 100ml and can go to 2000ml. </li></ul><ul><li>MAINTAIN QUALITY OF CELLS </li></ul><ul><li>Lack of bubbles and mechanical devices </li></ul><ul><li>SCALABLE TO 500 LITERS </li></ul>
  61. 62. Advantage of Wave Bioreactor <ul><li>COMPLETELY CLOSED SYSTEM Ideal for cell culture, GMP operations. </li></ul><ul><li>OPERATES WITH OR WITHOUT AN INCUBATOR </li></ul><ul><li>PROVEN FOR GMP OPERATIONS Used in the GMP production of human therapeutics. Closed system is easy to validate. All contact materials are FDA approved. </li></ul><ul><li>PERFUSION CULTURE OPTION Patented internal perfusion filters enable perfusion of media for high-density cell culture. </li></ul><ul><li>EASY TO OPERATE No complex piping or sterilization sequences. Simply place a new presterile Cellbag on the rocker; fill with media, and add your cells </li></ul>
  62. 63. Wave Bioreactor in Perfusion Mode
  63. 64. Packed-bed and fluidized-bed biofilm or immobilized-cell bioreactor
  64. 65. Tissue culture flasks (T-flasks)
  65. 66. Hollow Fiber Bioreactor
  66. 67. Hollow Fiber Bioreactor <ul><li>Intraluminal (Cells inside fibers ) </li></ul><ul><li>Extraluminal (Cells outside fibers) </li></ul><ul><li>Fibers are made of a porous material (PTFE and others). </li></ul><ul><li>Permits movement of small molecules (O2, glucose), but not cells </li></ul>
  67. 68. Cell Culture Systems <ul><li>Various cell culture systems were developed over a period of time </li></ul><ul><li>Small scale culture systems </li></ul><ul><ul><li>T-Flask </li></ul></ul><ul><ul><li>Spinners </li></ul></ul><ul><li>Large/production scale culture systems </li></ul><ul><ul><li>Roller bottle </li></ul></ul><ul><ul><li>Multiple plate culture systems </li></ul></ul><ul><ul><li>Bioreactors </li></ul></ul><ul><li>Stirred tank reactors </li></ul><ul><li>Disposable bioreactors </li></ul><ul><li>Airlift bioreactors </li></ul><ul><li>Spin filter stirred tank </li></ul><ul><li>Stirred tank bioreactors are most widely used </li></ul>